Compositions and methods for treating or preventing inflammatory bowel disease, familial adenomatous polyposis and colon cancer

ABSTRACT

The present invention provides methods for preventing colon cancer by administering, to a patient in need thereof, a polymeric proanthocyanidin composition from a  Croton  species or  Calophyllum  species in an amount sufficient to prevent colon cancer, which composition inhibits COX-2. The present invention also provides methods for treating inflammation locally in the intestines comprising administering to a patient in need thereof, a polymeric proanthocyanidin composition from a  Croton  species or  Calophyllum  species in an amount sufficient to treat inflammation. In one embodiment, the polymeric proanthocyanidin compound is crofelemer. The present invention in alternative embodiments provides methods for treating pain locally in the intestines and treating inflammatory bowel diseases, such as Crohn&#39;s disease and ulcerative colitis, as well as for treating familial adenomatous polyposis (FAP).

This application is a divisional application of U.S. patent applicationSer. No. 11/741,797, filed Apr. 30, 2007, which claims benefit ofpriority to U.S. provisional patent application 60/797,075, filed May 1,2006. The entire contents of each of the aforementioned applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Approximately 17 million Americans take nonsteroidal anti-inflammatorydrugs (NSAIDS) daily for treatment of pain or inflammation. These drugsare nonselective inhibitors of the enzymes cyclooxygenase (COX) 1 and 2,which convert arachidonic acid to prostaglandins. Two COX-2 inhibitors,celecoxib and rofecoxib, were introduced in 1999 in the biggest druglaunch ever. To date, more than 6.6 million prescriptions have beenfilled. A third drug, meloxicam, which was recently introduced in theUnited States, has a high affinity for COX-2 but also inhibits COX-1 attherapeutic doses. However, several of these COX-2 inhibitors have beenrecalled due to their side effects of increasing adverse cardiovascularevents. Thus, there remains a need for COX-2 inhibitors that can beadministered such that the adverse cardiovascular effects are avoided.

There is a great deal of evidence that inhibitors of COX-2 are useful intreating colon cancer and inflammatory diseases of the colon. Forexample, it has been observed that inhibitors of COX-2 sensitize coloncancer cells to a form of apoptosis called TRAIL-induced apoptosis(Martin et al., 2005, Cancer Research 65:11447-11458). Further, resultspresented at the American Society of Clinical Oncology 2005 AnnualMeeting showed that patients with stage ITT colon cancer may benefitfrom aspirin as much as from surgery and standard chemotherapy alone.The study also found a similar result for COX-2 inhibitors celecoxib(CELEBREX®) and rofecoxib (VIOXX®). Moreover, the COX-2 inhibitorcelecoxib (CELEBREX®) has been demonstrated to reduce the number ofcolon polyps in patients with Familial Adenomatous Polyposis (FAP).Patients with FAP develop hundreds to thousands of precancerous polyps(adenomas) throughout the colon and rectum. Left untreated, nearly allFAP patients develop colorectal cancer by their 40s and 50s. The primarytreatment for FAP is surgical removal of most or all of the colon andrectum with subsequent surveillance of any remaining colorectal segment.Based on NIH-sponsored clinical results, the FDA approved celecoxib(CELEBREX®) as an adjunctive drug (an accessory or auxiliary agent) thatcould be added to the standard of care in people with FAP. There is aneed in the art to develop COX-2 inhibitors that remain active in thedigestive tract and are not systemically absorbed.

U.S. Pat. Nos. 5,211,944 and 5,494,661 to Tempesta disclose the use of aproanthocyanidin polymeric composition isolated from Croton spp. orCalophyllum spp. for the treatment of viral infections. Rozhon et al.,U.S. Patent Publication No. 2005/0019389, disclose the use of aproanthocyanidin polymeric composition isolated from Croton spp. orCalophyllum spp. for the treatment of secretory or traveler's diarrhea.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art for the present application.

SUMMARY OF THE INVENTION

The present invention relates to methods for preventing or treatinginflammation or other COX-2-related physiological effects locally in theintestines by administering a composition that is an extract or latexfrom a Croton species or Calophyllum species. An exemplary compositionis a polymeric proanthocyanidin composition from a Croton species orCalophyllum species. The present invention also relates to preventing ortreating inflammatory diseases and disorders of the intestines, such asCrohn's disease and ulcerative colitis by administering a compositionthat is an extract or latex from a Croton species or Calophyllumspecies, such as a polymeric proanthocyanidin composition. The presentinvention also relates to treating or preventing colon cancer byadministering a composition that is an extract or latex from a Crotonspecies or Calophyllum species, such as a polymeric proanthocyanidincomposition. The present invention also relates to treating orpreventing familial adenomatous polyposis (FAP), particularly preventingor reducing the incidence of polyps in subjects that have FAP, byadministering a composition that is an extract or latex from a Crotonspecies or Calophyllum species, such as a polymeric proanthocyanidincomposition.

The present invention is based, in part, on the discovery that acomposition that is an extract or latex from a Croton species orCalophyllum species, particularly a polymeric proanthocyanidincomposition, e.g., crofelemer, not only inhibits COX-2, but alsoselectively inhibits COX-2 over COX-1. Moreover, since a polymericproanthocyanidin composition from a Croton species or Calophyllumspecies, such as crofelemer, is not substantially absorbed into theblood stream when orally administered, the known adverse cardiovascularside effects of other selective COX-2 inhibitors are avoided and theagent has activity particularly in the intestines. Extracts or latexesthat are systemically absorbed can be modified by methods known in theart to make such not absorbable.

In a particular embodiment, the present invention is directed to amethod of treating or preventing inflammation locally in the intestinescomprising administering to a patient in need of such treatment, anamount of a composition that is an extract or latex from a Crotonspecies or Calophyllum species effective to treat or prevent theinflammation. In another embodiment, the present invention is directedto a method of treating or preventing an inflammatory bowel diseasecomprising administering to a patient in need of such treatment orprevention, an amount of a composition that is an extract or latex froma Croton species or Calophyllum species effective to treat or preventthe inflammatory bowel disease. Exemplary inflammatory bowel diseasesinclude, but are not limited to, Crohn's disease and ulcerative colitis.In yet another embodiment, the present invention is directed to a methodof treating or preventing colon cancer comprising administering to apatient in need of such prevention an amount of a composition that is anextract or latex from a Croton species or Calophyllum species effectiveto treat or prevent colon cancer. In yet another embodiment, the presentinvention is directed to a method of treating or preventing familialadenomatous polyposis (FAP) comprising administering to a patient inneed of such prevention an amount of a composition that is an extract orlatex from a Croton species or Calophyllum species effective to treat orprevent FAP.

In an alternative embodiment, the present invention is directed to amethod for treating pain locally in the intestines comprisingadministering to a patient in need of such treatment or prevention, anamount of a composition that is an extract or latex from a Crotonspecies or Calophyllum species effective to treat the pain in theintestines.

In another particular embodiment, the present invention is directed to amethod of treating or preventing inflammation locally in the intestinescomprising administering to a patient in need of such treatment, anamount of a polymeric proanthocyanidin composition from a Croton speciesor Calophyllum species effective to treat or prevent the inflammation.In another embodiment, the present invention is directed to a method oftreating or preventing an inflammatory bowel disease comprisingadministering to a patient in need of such treatment or prevention, anamount of a polymeric proanthocyanidin composition from a Croton speciesor Calophyllum species effective to treat or prevent the inflammatorybowel disease. Exemplary inflammatory bowel diseases include, but arenot limited to, Crohn's disease and ulcerative colitis. In yet anotherembodiment, the present invention is directed to a method of treating orpreventing colon cancer comprising administering to a patient in need ofsuch prevention an amount of a polymeric proanthocyanidin compositionfrom a Croton species or Calophyllum species effective to treat orprevent colon cancer. In yet another embodiment, the present inventionis directed to a method of treating or preventing familial adenomatouspolyposis (FAP) comprising administering to a patient in need of suchprevention an amount of a polymeric proanthocyanidin composition from aCroton species or Calophyllum species effective to treat or prevent FAP.

In an alternative embodiment, the present invention is directed to amethod for treating pain locally in the intestines comprisingadministering to a patient in need of such treatment or prevention, anamount of a polymeric proanthocyanidin composition from a Croton speciesor Calophyllum species effective to treat the pain in the intestines.

Exemplary polymeric proanthocyanidin compositions useful in the presentinvention are preferably isolated from a Croton species or Calophyllumspecies. For example, the composition can be a proanthocyanidin polymercomposition isolated from a Croton spp. or Calophyllum spp. by anymethod known in the art. For example, the proanthocyanidin polymercomposition may be isolated from a Croton spp. or Calophyllum spp. bythe method disclosed in Example 2, infra, or disclosed in U.S. Pat. No.5,211,944 or in Ubillas et al., 1994, Phytomedicine 1: 77-106 or U.S.Patent Publication No. US 2005/0019389. PCT application PCT/US00/02687,published as WO 00/47062, discloses a method of manufacturing aproanthocyanidin polymeric composition isolated from Croton spp. orCalophyllum spp. In preferred embodiment, the composition is entericprotected, e.g., enteric coated.

In one preferred embodiment, a proanthocyanidin polymer composition ofthe invention is crofelemer. Crofelemer (CAS 148465-45-6) is anoligomeric proanthocyanidin of varying chain lengths derived from theDragon's Blood of Croton lecheri of the family Euphorbiaceae. Crofelemerhas an average molecular weight of approximately 1900 daltons toapproximately 2700 daltons. The monomers comprising crofelemer comprisecatechin, epicatechin, gallocatechin, and epigallocatechin. The chainlength of crofelemer ranges from about 3 to about 30 units with anaverage chain length of about 8 units.

In preferred embodiments, the polymeric proanthocyanidin composition isorally administered. In certain other embodiments, the polymericproanthocyanidin composition is administered as a suppository or enema.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the Effect of Crofelemer 125 mg bid onStool Frequency in Females.

FIG. 2 is a graph illustrating the Effect of Crofelemer 125 mg bid onUrgency in Females.

FIG. 3 is a graph illustrating the Effects of Crofelemer on AdequateRelief of IBS Symptoms in Females.

FIG. 4 is a graph illustrating the Effect of Crofelemer on Pain Score inFemales.

FIG. 5 is a graph illustrating the Effect of Crofelemer on Percent ofPain Free Days in Females.

FIG. 6 is a graph illustrating the Effect of Crofelemer on Pain Score inFemales.

FIG. 7 is a schematic of Tablet Core Manufacturing Process.

FIG. 8 is a schematic of Tablet-Core Spray Coating Process.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for preventing or treatinginflammation locally in the intestines by administering a compositionthat is an extract or latex from a Croton species or Calophyllumspecies, e.g., a polymeric proanthocyanidin composition. The presentinvention also relates to methods of preventing or treating inflammatorydiseases and disorders of the intestines, such as Crohn's disease andulcerative colitis, by administering a composition that is an extract orlatex from a Croton species or Calophyllum species. The presentinvention also relates to treating or preventing colon cancer byadministering a composition that is an extract or latex from a Crotonspecies or Calophyllum species. The present invention also relates totreating or preventing familial adenomatous polyposis (FAP) byadministering a composition that is an extract or latex from a Crotonspecies or Calophyllum species, such as a polymeric proanthocyanidincomposition.

The present invention is based, in part, on the discovery that acomposition that is an extract or latex, such as a polymericproanthocyanidin composition, from a Croton species or Calophyllumspecies, e.g., crofelemer, not only inhibits COX-2, but also selectivelyinhibits COX-2 over COX-1. Moreover, since a polymeric proanthocyanidincomposition from a Croton species or Calophyllum species, such ascrofelemer, is not substantially absorbed into the blood stream whenorally administered, the known adverse cardiovascular side effects ofother selective COX-2 inhibitors are avoided and the agent retainsactivity in the intestines. Extracts or latexes that are systemicallyabsorbed can be modified by methods known in the art to make such notabsorbable.

In one preferred embodiment, the composition of the invention is aproanthocyanidin polymer composition, preferably an aqueous solubleproanthocyanidin polymer composition. In another preferred embodiment,the compositions of the present invention are not substantiallysystemically absorbed when administered orally.

Proanthocyanidins are a group of condensed tannins. Tannins are found ina wide variety of plants and are classified as either hydrolyzable orcondensed. Many plants used in traditional medicine as treatment orprophylaxis for diarrhea have been found to contain tannins andproanthocyanidins in particular (see, e.g., Yoshida et al., 1993,Phytochemistry 32:1033; Yoshida et al., 1992, Chem. Pharm. Bull.,40:1997; Tamaka et al., 1992, Chem. Pharm. Bull. 40:2092).

Proanthocyanidins are comprised of at least two or more monomer unitsthat may be of the same or different monomeric structure. The monomerunits (generally termed “leucoanthocyanidin”) are generally monomericflavonoids which include catechins, epicatechins, gallocatechins,galloepicatechins, flavanols, flavonols, and flavan-3,4-diols,leucocyanidins and anthocyanidins. Therefore, the polymer chains arebased on different structural units, which create a wide variation ofpolymeric proanthocyanidins and a large number of possible isomers(Hemingway et al., 1982, J.C.S. Perkin, 1:1217). Larger polymers of theflavonoid 3-ol units are predominant in most plants, and are found withaverage molecular weights above 2,000 daltons, containing 6 or moreunits (Newman et al., 1987, Mag. Res. Chem., 25:118).

Proanthocyanidin polymers are found in a wide variety of plants,particularly those with a woody habit of growth (e.g., Croton spp. andCalophyllum spp.). A number of different Croton tree species, includingCroton sakutaris, Croton gossypifolius, Croton palanostima, Crotonlechleri, Croton erythrochilus and Croton draconoides, found in SouthAmerica, produce a red viscous latex sap called Sangre de Drago or“Dragon's Blood”. This red, viscous latex is widely known for itsmedicinal properties. For example, U.S. Pat. No. 5,211,944 firstdescribed the isolation of an aqueous soluble proanthocyanidin polymercomposition from Croton spp. and the use of the composition as anantiviral agent (See also Ubillas et al., 1994, Phytomedicine, 1:77).The proanthocyanidin polymer composition was shown to have antiviralactivity against a variety of viruses including, respiratory syncytial,influenza, parainfluenza and herpes viruses. U.S. Pat. No. 5,211,944also discloses the isolation of an aqueous soluble proanthocyanidinpolymer composition from Calophyllum inophylum and the use of thiscomposition as an antiviral agent.

Exemplary proanthocyanidin polymer compositions useful in the presentinvention are preferably isolated from a Croton spp. or Calophyllum sppby any method known in the art. For example, the proanthocyanidinpolymer composition may be isolated from a Croton spp. or Calophyllumspp. by the method disclosed in Example 2, infra, or disclosed in U.S.Pat. No. 5,211,944 or in Ubillas et al., 1994, Phytomedicine 1: 77-106.PCT application PCT/US00/02687, published as WO 00/47062, discloses amethod of manufacturing a proanthocyanidin polymeric compositionisolated from Croton spp. or Calophyllum spp.

In one preferred embodiment, a proanthocyanidin polymer composition ofthe invention is crofelemer. Crofelemer (CAS 148465-45-6) is anoligomeric proanthocyanidin of varying chain lengths derived from theDragon's Blood of Croton lecheri of the family Euphorbiaceae. Crofelemerhas an average molecular weight of approximately 1900 daltons toapproximately 2700 daltons. The monomers comprising crofelemer comprisecatechin, epicatechin, gallocatechin, and epigallocatechin. The chainlength of crofelemer ranges from about 3 to about 30 units with anaverage chain length of about 8 units. The structure of crofelemer isshown below.

-   -   R═H or OH    -   n=3-30        wherein the average n=6.

Another illustrative method for isolating crofelemer can be found inU.S. Patent Publication No. 2005/0019389, the contents of which areexpressly incorporated herein. Purity of isolated crofelemer may vary.For example, purity of crofelemer may be above 70%, above 80%, above90%, up to essentially 100% purity, including all values in between,such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and greater than 99%purity.

In other embodiments of the invention, the composition useful in themethods of the invention is a raw latex obtained from a Croton speciesor a Calophyllum species or an extract obtained from a Croton species ora Calophyllum species that are not specifically polymericproanthocyanidin compositions. Exemplary extracts are described inPersinos et al., 1979, J. Pharma. Sci. 68:124 and Sethi, 1977, CanadianJ. Pharm. Sci. 12:7.

In a preferred embodiment, the compositions used in the methods of theinvention are not substantially systemically absorbed when administeredorally, i.e., only 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less than0.5% absorbed of the dosage given.

The present invention encompasses methods for treating and/or preventinginflammation locally in the intestines, treating and/or preventinginflammatory bowel disease and/or treating or preventing colon cancer,treating or preventing FAP, as well as treating pain locally in theintestines, in warm blooded animals, including male and female humans.The methods of the invention generally comprise administering to asubject in need of treatment a polymeric proanthocyanidin composition inaccordance with the invention. In a preferred embodiment, thecomposition is orally administered and is not systemically absorbed. Inanother embodiment, the composition is enteric protected. In anotherembodiment, the composition is rectally administered as a suppository.

In a particular embodiment, the present invention is directed to amethod of treating or preventing inflammation locally in the intestinescomprising administering to a patient in need of such treatment, anamount of a polymeric proanthocyanidin composition from a Croton speciesor Calophyllum species effective to treat or prevent the inflammation.The treatment, i.e., reduction, of inflammation in the intestines can bemeasured using any assay known in the art or available to the skilledphysician to measure inflammation including, but not limited to, assaysbased on biopsied tissue as well as visual assays, e.g., colonoscopy. Incertain embodiments, the level of inflammation has been decreased by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 809%,90%, or by at least 100% as compared to before treatment.

In another embodiment, the present invention is directed to a method oftreating or preventing an inflammatory bowel disease comprisingadministering to a patient in need of such treatment or prevention, anamount of a polymeric proanthocyanidin composition from a Croton speciesor Calophyllum species effective to treat or prevent the inflammatorybowel disease. Exemplary inflammatory bowel diseases include, but arenot limited to, Crohn's disease and ulcerative colitis.

In yet another embodiment, the present invention is directed to a methodof treating or preventing colon cancer comprising administering to apatient in need of such prevention an amount of a polymericproanthocyanidin composition from a Croton species or Calophyllumspecies effective to treat or prevent colon cancer. For example, apatient in need of such treatment or prevention includes those patientswho have been identified to have colon polyps, or those patients whohave been diagnosed with colon cancer (at any of the stages, i.e., Stage0, I, II, III, or IV), or those patients who have been treatedpreviously for colon cancer (e.g., to prevent recurrence), or thosepatients who have a familial or genetic predisposition to colon cancer,or those patients living in areas that have higher than average rates ofcolon cancer. Other patients in need of such treatment or prevention arethose who have had a colon biopsy indicating pre-cancerous changes.

In certain embodiments, the polymeric proanthocyanidin compositions ofthe invention can be administered as adjuvant therapy with other knowntherapies for treating or preventing colon cancer. For example, thecompositions of the invention can be administered before, concurrentlywith or after surgery, radiation therapy, chemotherapy or biologictherapy (biologic therapy includes immunologic therapy with engineeredantibodies such as ERBITUX(R, AVASTIN®, or a therapy to boost the immuneresponse to the cancer).

In an another embodiment, the present invention is directed to a methodfor treating pain locally in the intestines comprising administering toa patient in need of such treatment or prevention, an amount of apolymeric proanthocyanidin composition from a Croton species orCalophyllum species effective to treat the pain in the intestines.

Pain and discomfort can be measured by any method known in the art, forinstance on a pain or discomfort scale in which a patient assigns thelevel of pain or discomfort on a scale of 0 to 5, with 0 being no painor discomfort and 5 being assigned the highest level of pain ordiscomfort. In certain embodiments, the alleviation of pain ordiscomfort is measured by a lowering of the average level of pain ordiscomfort, an increase in the number of pain- or discomfort-free days.In certain embodiments, the number of pain- or discomfort-free days isincreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or byat least 50% compared to before treatment. In other embodiments, thelevel of pain or discomfort is decreased by at least 0.1, 0.2, 0.3, 0.4or by at least 0.5 units compared to before treatment, or even by atleast 1 unit, 1.5 units, or 2 units.

The compositions of the invention can be administered in a single or adivided dosage from one, two, three or four times per day. In aparticular embodiment, the composition is administered twice daily. Thepolymeric proanthocyanidin compositions of the present invention arepreferably administered orally. However, in certain embodiments, thecompositions can be administered rectally, either as a suppository or asan enema.

In certain preferred embodiments of the present invention, the polymericproanthocyanidin composition is crofelemer (CAS 148465-45-6). In otherpreferred aspects, crofelemer is administered orally. In yet otherpreferred embodiments, the composition is formulated so as to protectthe composition from the stomach environment, i.e., from the acidicenvironment and digestive proteins found in the stomach. In a preferredembodiment, administration is by oral route and the composition isenteric protected crofelemer. In another embodiment, the composition ofthe invention is formulated as a controlled release formulation.

When used according to the formulations and methods of the presentinvention, effective dosage ranges of the pharmaceutical formulations ofthe proanthocyanidin polymer composition for oral administration are inthe range of 0.1 to 100 mg/kg per day, preferably about 0.1 to about 40mg/kg per day, optionally 0.1 to 25 mg/kg per day, and also optionally0.1 to 10 mg/kg per day. It should be appreciated that the appropriatedose will depend upon the type and severity of the inflammatory boweldisease or pain. It has been found that human subjects can tolerate atleast up to 2 grams of the proanthocyanidin polymer composition per day(25-30 mg/kg/day) for up to 2 days. It is believed that doses may exceed40 mg/kg per day, optionally up to 100 mg/kg per day, if such dosagesare necessary.

In certain preferred embodiments, crofelemer is orally administered inan enteric protected form (enteric coated) in a total amount of not lessthan about 50 mg/day. As used herein, about means within the margin oferror. In specific embodiments, the enteric coated crofelemer is orallyadministered in an amount of from about 50 mg/day to 4000 mg/day. Inother specific embodiments, the enteric coated crofelemer is orallyadministered in an amount from about 100 mg/day to 2000 mg/day. Inanother embodiment, the enteric coated crofelemer is orally administeredto a subject in a total amount of not less than about 500 mg/day. Inspecific embodiments, the enteric coated crofelemer is orallyadministered to a subject in an amount of from about 250 mg/day to about2000 mg/day. In other embodiments, the enteric coated crofelemer isorally administered to a subject at not less than about 1750 mg/day,about 1700 mg/day, about 1650 mg/day, about 1600 mg/day, about 1550mg/day, about 1500 mg/day, about 1450 mg/day, about 1400 mg/day, about1350 mg/day, about 1300 mg/day, about 1250 mg/day, 1200 mg/day, about1150 mg/day, about 1100 mg/day, about 1050 mg/day, about 1000 mg/day,about 950 mg/day, about 900 mg/day, about 850 mg/day, about 800 mg/day,about 750 mg/day, about 700 mg/day, 650 mg/day, about 600 mg/day, about550 mg/day, about 500 mg/day, about 450 mg/day, about 400 mg/day, about350 mg/day, about 300 mg/day, or about 250 mg/day of orally administeredenteric coated crofelemer. In yet another embodiment, the enteric coatedcrofelemer is orally administered to a subject in an amount from about500 mg/day to 1000 mg/day. In other embodiments, the enteric coatedcrofelemer is orally administered to a subject in an amount of fromabout 250 mg/day to about 2000 mg/day, from about 250 mg/day to about1000 mg/day, from about 250 mg/day to about 750 mg/day, from about 250mg/day to about 500 mg/day, from about 350 mg/day to about 650 mg/day,or from about 500 mg/day to about 750 mg/day. In other particularembodiments, the total dosage of the enteric coated crofelemer orallyadministered to a subject is not less than about 50 mg, about 55 mg,about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185mg, about 190 mg, about 195 mg, about 200 mg, about 205 mg, about 210mg, about 215 mg, about 220 mg, about 225 mg, about 230 mg, about 235mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310mg, about 315 mg, about 320 mg, about 325 mg, about 330 mg, about 335mg, about 340 mg, about 345 mg, about 350 mg, about 355 mg, about 360mg, about 365 mg, about 370 mg, about 375 mg, about 380 mg, about 385mg, about 390 mg, about 395 mg, about 400 mg, about 405 mg, about 410mg, about 415 mg, about 420 mg, about 425 mg, about 430 mg, about 435mg, about 440 mg, about 445 mg, about 450 mg, about 455 mg, about 460mg, about 465 mg, about 470 mg, about 475 mg, about 480 mg, about 485mg, about 490 mg, about 495 mg, about 500 mg, about 505 mg, about 510mg, about 515 mg, about 520 mg, about 525 mg, about 530 mg, about 535mg, about 540 mg, about 545 mg, about 550 mg, about 555 mg, about 560mg, about 565 mg, about 570 mg, about 575 mg, about 580 mg, about 585mg, about 590 mg, about 595 mg, about 600 mg, about 605 mg, about 610mg, about 615 mg, about 620 mg, about 625 mg, about 630 mg, about 635mg, about 640 mg, about 645 mg, about 650 mg, about 655 mg, about 660mg, about 665 mg, about 670 mg, about 675 mg, about 680 mg, about 685mg, about 690 mg, about 695 mg, about 700 mg, about 705 mg, about 710mg, about 715 mg, about 720 mg, about 725 mg, about 730 mg, about 735mg, about 740 mg, about 745 mg, about 750 mg, about 755 mg, about 760mg, about 765 mg, about 770 mg, about 775 mg, about 780 mg, about 785mg, about 790 mg, about 795 mg, about 800 mg, about 805 mg, about 810mg, about 815 mg, about 820 mg, about 825 mg, about 830 mg, about 835mg, about 840 mg, about 845 mg, about 850 mg, about 855 mg, about 860mg, about 865 mg, about 870 mg, about 875 mg, about 880 mg, about 885mg, about 890 mg, about 895 mg, about 900 mg, about 905 mg, about 910mg, about 915 mg, about 920 mg, about 925 mg, about 930 mg, about 935mg, about 940 mg, about 945 mg, about 950 mg, about 955 mg, about 960mg, about 965 mg, about 970 mg, about 975 mg, about 980 mg, about 985mg, about 990 mg, about 995 mg, or not less than about 1000 mg, once,twice, or three-times per day.

In other embodiments of the invention, the composition, theproanthocyanidin polymer composition is preferably given at a dosagethat is bioequivalent to orally administered enteric coated crofelemerat a dosage of about 50 mg per day to about 4000 mg/day or any of thedoses listed above. In one embodiment, bioequivalency is a sufficientdose of a composition of the invention to produce a similar therapeuticeffect as seen with another composition at a particular dosage, e.g.,enteric coated crofelemer at a dosage of about 50 mg per day to about4000 mg per day. In another embodiment, bioequivalency is as defined by,or is as determined in accordance with methods approved by, the U.S.Food and Drug Administration.

In a preferred embodiment, crofelemer is enteric coated so as to protectit from degradation by the acidic conditions of the stomach and/or frominteractions with proteins, such as pepsin, present in the stomach,i.e., an enteric protected formulation. In a specific embodiment,crofelemer is in tablet form. In yet another specific embodiment, thetablet is enteric coated, e.g., EUDRAGIT®. In a preferred embodiment,crofelemer is formulated as an enteric coated bead or granule in anenteric coated capsule shell. In another embodiment, crofelemer isformulated in a delayed release composition, e.g., Merck GEM or AlzaOROS (release is primarily delayed until after the formulation passesout of the stomach and into the intestine).

In certain embodiments, the composition is formulated with a compound orcompounds which neutralize stomach acid. Alternatively, thepharmaceutical composition containing the composition is administeredeither concurrent with or subsequent to or after administration of apharmaceutical composition which neutralize stomach acid. Compounds,such as antacids, which are useful for neutralizing stomach acidinclude, but are not limited to, aluminum carbonate, aluminum hydroxide,bismuth subnitrate, bismuth subsalicylate, calcium carbonate,dihydroxyaluminum sodium carbonate, magaldrate, magnesium carbonate,magnesium hydroxide, magnesium oxide, and mixtures thereof. Compoundsthat are able to reduce the secretion of stomach acid and/or are able toreduce the acidity of stomach fluid are well known in the art andinclude, but are not limited to, antacids (aluminum hydroxide, aluminumcarbonate, aluminum glycinate, magnesium oxide, magnesium hydroxide,magnesium carbonate, calcium carbonate, sodium bicarbonate), stomachacid blockers (cimetidine (TAGAMET™), famotidine (MYLANTA™, PEPCID™),nizatidine (AXID™), ranitidine (ZANTAC™) and omeprazole (ZEGERID™)) anda combination of any of the foregoing. In general, any drug that hasbeen approved for sale by the relevant government agency and is able toreduce the production of stomach acid and/or reduce the acidity ofstomach fluid can be administered in combination with an inhibitormolecule, such as crofelemer, in accordance with the methods of theinvention.

In a particular embodiment where crofelemer is not enteric coated,crofelemer is formulated with one or more compounds that are able toreduce the secretion of stomach acid and/or able to reduce the acidityof stomach fluid. Preferably, the dosage of crofelemer to be given inthis formulation is a dosage that is bioequivalent to orallyadministered enteric coated crofelemer at a dosage of about 50 mg perday to about 4000 mg per day. In an exemplary embodiment, crofelemer isformulated in a controlled release (delayed release) composition, suchas Merck GEM, Alza OROS, wax matrix (release is primarily delayed untilafter the formulation passes out of the stomach and into the intestine).

In other embodiments, the compositions of the invention can beadministered in combination with analgesic or anti-inflammatory agents,if necessary. In a preferred embodiment, the analgesic oranti-inflammatory agent is formulated or modified such that it is notsubstantially systemically absorbed, i.e., only 20%, 15%, 10%, 5%, 4%,3%, 2%, 1% or 0.5% absorbed of the dosage given.

The present invention also provides pharmaceutical formulations of thepolymeric proanthocyanidin compositions of the invention comprising thecomposition along with a pharmaceutically acceptable vehicle, at a dosewhich is therapeutically effective at treating inflammation, pain or aninflammatory bowel disease. In one embodiment, a directly compressibleproanthocyanidin polymer composition (i.e., that can be directlycompressed, without excipients, into a tablet of pharmaceuticallyacceptable hardness and friability) compressed into a tablet, optionallywith a lubricant, such as but not limited to magnesium stearate, isenteric coated. In another embodiment, the pharmaceutical compositionsof the invention alternatively include one or more substances thateither neutralize stomach acid and/or enzymes or are active to preventsecretion of stomach acid. These formulations can be prepared by methodsknown in the art, see, e.g., methods described in Remington'sPharmaceutical Sciences, 18th Ed., ed. Alfonso R. Gennaro, MackPublishing Co., Easton, Pa., 1990.

In another preferred embodiment, the pharmaceutical compositioncomprises a proanthocyanidin polymer composition prepared from a Crotonspp, the dosage of which is a bioequivalent dosage of orallyadministered enteric protected crofelemer of 50 mg per day, preferably500 mg/day. In a preferred embodiment, the proanthocyanidin polymercomposition of the present invention is crofelemer (CAS 148465-45-6).

The compositions of the invention can be provided in any therapeuticallyacceptable pharmaceutical form. The pharmaceutical composition can beformulated for oral administration as, for example but not limited to,drug powders, crystals, granules, small particles (which includeparticles sized on the order of micrometers, such as microspheres andmicrocapsules), particles (which include particles sized on the order ofmillimeters), beads, microbeads, pellets, pills, microtablets,compressed tablets or tablet triturates, molded tablets or tablettriturates, and in capsules, which are either hard or soft and containthe composition as a powder, particle, bead, solution or suspension. Thepharmaceutical composition can also be formulated for oraladministration as a solution or suspension in an aqueous liquid, as aliquid incorporated into a gel capsule or as any other convenientformulation for administration, or for rectal administration, as asuppository, enema or other convenient form. The compositions of theinvention can also be provided in a controlled release formulation, suchas Merck GEM, Alza OROS, wax matrix.

The pharmaceutical formulation can also include any type ofpharmaceutically acceptable excipients, additives or vehicles. Forexample, but not by way of limitation, diluents or fillers, such asdextrates, dicalcium phosphate, calcium sulfate, lactose, cellulose,kaolin, mannitol, sodium chloride, dry starch, sorbitol, sucrose,inositol, powdered sugar, bentonite, microcrystalline cellulose, orhydroxypropylmethylcellulose may be added to the inhibitor molecule toincrease the bulk of the composition. Also, binders, such as but notlimited to, starch, gelatin, sucrose, glucose, dextrose, molasses,lactose, acacia gum, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapgol husks, carboxymethylcellulose,methylcellulose, polyvinylpyrrolidone, Veegum and starch arabogalactan,polyethylene glycol, ethylcellulose, and waxes, may be added to theformulation to increase its cohesive qualities. Additionally,lubricants, such as but not limited to, talc, magnesium stearate,calcium stearate, stearic acid, hydrogenated vegetable oils,polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride,leucine, carbowax, sodium lauryl sulfate, and magnesium lauryl sulfatemay be added to the formulation. Also, glidants, such as but not limitedto, colloidal silicon dioxide or talc may be added to improve the flowcharacteristics of a powdered formulation. Finally, disintegrants, suchas but not limited to, starches, clays, celluloses, algins, gums,crosslinked polymers (e.g., croscarmelose, crospovidone, and sodiumstarch glycolate), Veegum, methylcellulose, agar, bentonite, celluloseand wood products, natural sponge, cation-exchange resins, alginic acid,guar gum, citrus pulp, carboxymethylcellulose, or sodium lauryl sulfatewith starch may also be added to facilitate disintegration of theformulation in the intestine.

In one aspect of this embodiment, crofelemer is formulated for oraladministration. In other aspects, the pharmaceutical dosage form isformulated to protect the composition, e.g., crofelemer, fromdegradation by the acidic conditions of the stomach and frominteractions with proteins, such as pepsin, present in the stomach.Thus, in a preferred aspect, the formulation is enteric coated. Forexample, the enteric coated formulation is enteric coated tablets, beadsor granules, which optionally contain a lubricant such as, but notlimited to, magnesium stearate. The enteric coated formulations includeenteric coated beads in a capsule, enteric coated microspheres in acapsule, enteric coated microspheres provided in a suspension or mixedwith food, which suspensions are particularly convenient for pediatricadministration, and enteric coated compressed tablets. The capsule canbe a hard-shell gelatin capsule or a cellulose capsule. In particular,the pharmaceutical composition is formulated as an enteric coatedcapsule. In one specific aspect, a proanthocyanidin polymer compositionis administered in tablet form, which tablet is backfilled withmicrocrystalline cellulose.

In one embodiment, the composition is directly compressed, that is, thecomposition of the invention, with or without any excipients, can becompressed into a tablet, or other pharmaceutical formulation, that hasa pharmaceutically acceptable hardness and friability. Preferably, thedirectly compressible pharmaceutical composition can be compressed intotablets having a hardness of greater than 4 kp (kiloponds), preferably ahardness of 8 to 14 kp, more preferably a hardness of 10 to 13 kp. Adirectly compressible composition can be compressed into a tablet thathas a friability of not more than 1% loss in weight, preferably lessthan 0.8% loss in weight, more preferably less than 0.5% loss in weight.

In a preferred embodiment, the directly compressible formulationconsists of 99.93% crofelemer and 0.07% magnesium stearate and is coatedwith a methacrylic acid copolymer. In another preferred embodiment, thepharmaceutical formulation contains a directly compressible compositionbut no excipients, additives or vehicles other than an enteric coating;however, the formulation may contain a lubricant, such as but notlimited to, magnesium stearate. Preferably, a directly compressedproanthocyanidin polymer composition formulation is formulated as atablet of pharmaceutically acceptable hardness (greater than 4 kp,preferably 8-14 kp, and more preferably 10-13 kp) and friability (notmore than 1% loss in weight, preferably less than 0.8% loss in weight,and more preferably less than 0.5% loss in weight).

In a more preferred embodiment, a composition of the invention isenteric coated. Enteric coatings are those coatings that remain intactin the stomach, but will dissolve and release the contents of the dosageform once it reaches the small intestine. A large number of entericcoatings are prepared with ingredients that have acidic groups suchthat, at the very low pH present in the stomach, i.e. pH 1.5 to 2.5, theacidic groups are not ionized and the coating remains in anundissociated, insoluble form. At higher pH levels, such as in theenvironment of the intestine, the enteric coating is converted to anionized form, which can be dissolved to release the inhibitor molecule.Other enteric coatings remain intact until they are degraded by enzymesin the small intestine, and others break apart after a defined exposureto moisture, such that the coatings remain intact until after passageinto the small intestines.

Polymers which are useful for the preparation of enteric coatingsinclude, but are not limited to, shellac, starch and amylose acetatephthalates, styrene-maleic acid copolymers, cellulose acetate succinate,cellulose acetate phthalate (CAP), polyvinylacctatc phthalatc (PVAP),hydroxypropylmcthylccllulosc phthalatc (grades HP-50 and HP-55),ethylcellulose, fats, butyl stearate, and methacrylic acid-methacrylicacid ester copolymers with acid ionizable groups (including “ACRYLEZE®”and “EUDRAGIT®”), such as “EUDRAGITR L 30D”, “EUDRAGIT® RL 30D”,“EUDRAGIT® RS 30D”, “EUDRAGIT® L 100-55”, and “EUDRAGIT® L 30D-55”. In apreferred embodiment, the pharmaceutical composition contains apolymeric proanthocyanidin composition and the enteric coating polymer“EUDRAGIT® L 30D”, an anionic copolymer of methacrylic acid and methylacrylate with a mean molecular weight of 250,000 Daltons. In anotherpreferred embodiment, the enteric coating polymer is “EUDRAGIT® L30D-55”.

The disintegration of the enteric coating occurs either by hydrolysis byintestinal enzymes or by emulsification and dispersion by bile salts,depending upon the type of coating used. For example, esteraseshydrolyze esterbutyl stearate to butanol and stearic acid and, as thebutanol dissolves, the stearic acid flakes off of the medicament.Additionally, bile salts emulsify and disperse ethylcellulose,hydroxypropylmethylcellulose, fats and fatty derivatives. Other types ofcoatings are removed depending on the time of contact with moisture, forexample coatings prepared from powdered carnauba wax, stearic acid, andvegetable fibers of agar and elm bark rupture after the vegetable fibersabsorb moisture and swell. The time required for disintegration dependsupon the thickness of the coating and the ratio of vegetable fibers towax.

Application of the enteric coating to the polymeric proanthocyanidincomposition of the invention can be accomplished by any method known inthe art for applying enteric coatings. For example, but not by way oflimitation, the enteric polymers can be applied using organic solventbased solutions containing from 5 to 10% w/w polymer for sprayapplications and up to 30% w/w polymer for pan coatings. Solvents thatare commonly in use include, but are not limited to, acetone,acetone/ethyl acetate mixtures, methylene chloride/methanol mixtures,and tertiary mixtures containing these solvents. Some enteric polymers,such as methacrylic acid-methacrylic acid ester copolymers can beapplied using water as a dispersant. The volatility of the solventsystem must be tailored to prevent sticking due to tackiness and toprevent high porosity of the coating due to premature spray drying orprecipitation of the polymer as the solvent evaporates.

Furthermore, plasticizers can be added to the enteric coating to preventcracking of the coating film. Suitable plasticizers include the lowmolecular weight phthalate esters, such as diethyl phthalate, acetylatedmonoglycerides, triethyl citrate, polyethyl glycoltributyl citrate andtriacetin. Generally, plasticizers are added at a concentration of 10%by weight of enteric coating polymer weight. Other additives such asemulsifiers, for example detergents and simethicone, and powders, forexample talc, may be added to the coating to improve the strength andsmoothness of the coating. Additionally, pigments may be added to thecoating to add color to the pharmaceutical formulation.

In preferred embodiments, a pharmaceutical composition of the polymericproanthocyanidin composition is provided as enteric coated beads inhard-shell gelatin capsules. In a preferred embodiment, proanthocyanidinpolymer beads are prepared by mixing a proanthocyanidin polymercomposition with hydroxypropylmethylcellulose and layering the mixtureonto nonpareil seeds (sugar spheres). In a more preferred embodiment,crofelemer, which is directly compressible, without any excipients,additives or vehicles other than an enteric coating, is milled andfractionated into beads (i.e., as beads that do not contain thenonpareil sugar seeds). The beads may be covered with a seal coat ofOpadry Clear (mixed with water). A preferred enteric coating of thebeads is “EUDRAGIT™ L 30D” or “EUDRAGIT™ L 30D-55” applied as an aqueousdispersion containing 20%-30% w/w dry polymer substance, which issupplied with 0.7% sodium lauryl sulfate NF (SLS) and 2.3% polysorbate80 NF (Tween™ 20) as emulsifiers, to which plasticizers, such aspolyethylene glycol and/or citric acid esters, are added to improve theelasticity of the coating, and talc can be added to reduce the tendencyof the enteric coating polymer to agglutinate during the applicationprocess and to increase the smoothness of the film coating.

In a preferred formulation, the final composition of enteric coatedproanthocyanidin polymer composition beads containing the nonpareilseeds is 17.3% w/w nonpareil seeds, 64.5% w/w proanthocyanidin polymercomposition, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadryclear, 14.5% w/w “EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate, and0.25% w/w glyceryl monostearate. This pharmaceutical formulation may beprepared by any method known in the art or by the method described inExample 1, infra.

A preferred formulation of the proanthocyanidin polymer compositionbeads not containing the nonpareil seeds is 78% w/w directlycompressible proanthocyanidin polymer composition (e.g., isolated by themethod described in the Examples), 0.76% w/w Opadry Clear, 19% w/w“EUDRAGIT™ L 30D-55”, 1.9% triethyl citrate, and 0.34% w/w glycerylmonostearate. This pharmaceutical formulation may be prepared by anymethod known in the art or by the method described in Example 2, infra.

Another preferred formulation contains 54.58% w/w proanthocyanidinpolymer composition beads (without non-pareil seeds and made of adirectly compressible proanthocyanidin polymer composition), 1.78% w/wOpadry Clear, 39% w/w “EUDRAGIT™ L 30D-55”, 3.9% triethylcitrate, and0.74% w/w glyceryl monostearate.

In another embodiment, the pharmaceutical composition comprising thepolymeric proanthocyanidin composition of the invention is formulated asenteric coated granules or powder (microspheres with a diameter of300-500μ) provided in either hard shell gelatin capsules or suspended inan oral solution for pediatric administration. The enteric coated powderor granules may also be mixed with food, particularly for pediatricadministration. This preparation may be prepared using techniques wellknown in the art, such as the method described in Example 1 C, infra.

In general, the granules and powder can be prepared using any methodknown in the art, such as but not limited to, crystallization,spray-drying or any method of comminution, preferably using a high speedmixer/granulator. Examples of high speed mixer/granulators include the“LITTLEFORD LODIGE™” mixer, the “LITTLEFORD LODIGE™” MGTmixer/granulator, and the “GRAL™” mixer/granulator. During thehigh-shear powder mixing, solutions of granulating agents, calledbinders, are sprayed onto the powder to cause the powder particles toagglomerate, thus forming larger particles or granules. Granulatingagents which are useful for preparing the granules, include but are notlimited to, cellulose derivatives (including carboxymethylcellulose,methylcellulose, and ethylcellulose), gelatin, glucose,polyvinylpyrrolidone (PVP), starch paste, sorbitol, sucrose, dextrose,molasses, lactose, acacia gum, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks, Veegum and larcharabogalactan, polyethylene glycol, and waxes. Granulating agents may beadded in concentrations ranging from 1 to 30% of the mass of theparticles or granules.

The powder or granules are preferably coated using the fluidized bedequipment. The granules or powder may then be covered with a seal coatof Opadry Clear (mixed with water). A preferred enteric coating is“EUDRAGIT™ L 30D” applied as an aqueous dispersion containing 30% w/wdry polymer substance, which is supplied with 0.7% sodium lauryl sulfateNF (SLS) and 2.3% polysorbate 80 NF (Tween™ 20) as emulsifiers, to whichthe plasticizers, polyethylene glycol and citric acid esters, are addedto improve the elasticity of the coating, and talc is added to reducethe tendency of the enteric coating polymer to agglutinate during theapplication process and to increase the smoothness of the film coating.In a preferred embodiment, the final composition of an enteric coatedpowder is 81.8% w/w proanthocyanidin polymer composition, 1.5% w/whydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% w/w“EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate, and 0.25% w/w glycerylmonostearate. The final composition of the enteric coated granules is81.8% w/w proanthocyanidin polymer composition, 10%polyvinylpyrrolidone, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/wOpadry clear, 14.5% w/w “EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate,and 0.25% w/w glyceryl monostearate.

The enteric coated granules or powder particles can further be suspendedin a solution for oral administration, particularly for pediatricadministration. The suspension can be prepared from aqueous solutions towhich thickeners and protective colloids are added to increase theviscosity of the solution to prevent rapid sedimentation of the coatedpowder particles or granules. Any material which increases the strengthof the hydration layer formed around suspended particles throughmolecular interactions and which is pharmaceutically compatible with theinhibitor molecule can be used as a thickener, such as but not limitedto, gelatin, natural gums (e.g., tragacanth, xanthan, guar, acacia,panwar, ghatti, etc.), and cellulose derivatives (e.g., sodiumcarboxymethylcellulose, hydroxypropylcellulose, andhydroxypropylmethylcellulose, etc.). Optionally, a surfactant such asTween™ may be added to improve the action of the thickening agent. Apreferred suspension solution is a 2% w/w hydroxypropylmethylcellulosesolution in water containing 0.2% Tween™.

The polymeric proanthocyanidin composition can also be formulated asenteric coated tablets. In one preferred embodiment, a proanthocyanidinpolymer composition is granulated with any pharmaceutically acceptablediluent (such as those listed above) by the methods described above forpreparing the granules. Then, the granules are compressed into tabletsusing any method well known in the art, for example but not limited to,the wet granulation method, the dry granulation method or the directcompression method. Preferred diluents include, but are not limited to,microcrystalline cellulose (“AVICEL™ PH 200/300”) and dextrates(“EMDEX™”). Additionally, disintegrants, such as those described above,and lubricants, such those described above, may also be added to thetablet formulation. A preferred tablet formulation contains 250 mgproanthocyanidin polymer composition, 7 mg of the disintegrant“AC-DI-SOL™” (cross linked sodium carboxymethylcellulose), 1.75 mg ofthe lubricant magnesium stearate and the weight of “AVICEL™ PH 200/300”necessary to bring the mixture up to 350 mg. The tablets are coated withan enteric coating mixture prepared from 250 grams “EUDRAGIT™ L 30D-55”, 7.5 grams triethyl citrate, 37.5 grams talc and 205 grams water.This formulation may be prepared by any method well known in the art.

In a preferred embodiment, a directly compressible proanthocyanidinpolymer composition is made into granules by size reduction (e.g., asdescribed above) and mixed with a lubricant, preferably, magnesiumstearate. Then, the lubricated granules are compressed into tabletsusing any method well-known in the art, for example but not limited to,the direct compression method. Preferably, each tablet is 125 mgcontaining 99.6% w/w directly compressible proanthocyanidin polymercomposition and 0.40% w/w magnesium stearate. The tablets are thenpreferably coated with an enteric coating mixture of a 30% suspension(6.66 g in 22.22 g) of “EUDRAGIT™ L 30D-55”, 0.67 g triethyl citrate,1.67 g talc and 20.44 g purified water, per 100 grams of tablet. Thetablets can be prepared by any method known in the art or by the methoddescribed in Example 1E, infra.

In another embodiment, a directly compressible proanthocyanidin polymercomposition is formulated into core tablets of either 125 mg, 250 mg or500 mg containing 99.6% w/w directly compressible proanthocyanidinpolymer composition and 0.40% w/w magnesium stearate. The tablets arethen preferably coated with an enteric coating mixture. The finalcomposition of the tablets is 86.6% w/w directly compressibleproanthocyanidin polymer composition, 0.4% magnesium stearate, 6.5%“EUDRAGIT™ L30D-55”, 0.9% triethyl citrate, 2.87% talc, and 2.74% whitedispersion. The tablets can be prepared by any method known in the art,for example but not limited to, the method described infra.

The compositions formed into small particles (which include particlessized on the order of micrometers, such as microspheres andmicrocapsules), particles (which include particles sized on the order ofmillimeters), drug crystals, pellets, pills and microbeads can be coatedusing a fluidized-bed process. This process uses fluidized-bedequipment, such as that supplied by “GLATT™”, “AEROMATIC™”, “WURSTER™”,or others, by which the composition cores are whirled up in a closedcylindrical vessel by a stream of air, introduced from below, and theenteric coat is formed by spray drying it onto the cores during thefluidization time. To coat tablets or capsules, Accela-Cota coatingequipment (“MANESTY™”) can be used. By this process, the tablets orcapsules are placed in a rotating cylindrical coating pan with aperforated jacket and spraying units are installed within the pan andthe dry air is drawn in through the rotating tablets or capsules. Anyother type of coating pan, such as the “COMPU-LAB™” pan, Hi-coates“GLATT™” immersion sword process, the “DRIAM™” Dricoater, “STEINBERG™”equipment, “PELLEGRINI™” equipment, or “WALTHER™” equipment can also beused.

The pharmaceutical formulations of the invention can also be used totreat non-human animals, particularly in farm animals, such as but notlimited to, bovine animals, swine, ovine animals, poultry (such aschickens), and equine animals, and other domesticated animals such ascanine animals and feline animals. In particular, the pharmaceuticalformulations of the invention can be used to treat non-human animals,particularly food animals such as cattle, sheep and swine byincorporating the pharmaceutical compositions of the invention into theanimal's feed.

Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. The practice of the present invention willemploy, unless otherwise indicated, conventional techniques of molecularbiology (including recombinant techniques), microbiology, cell biology,biochemistry, nucleic acid chemistry, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature, such as, Current Protocols in Immunology (J. E. Coligan etal., eds., 1999, including supplements through 2001); Current Protocolsin Molecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); Molecular Cloning: A Laboratory Manual, thirdedition (Sambrook and Russel, 2001); PCR: The Polymerase Chain Reaction,(Mullis et al., eds., 1994); The Immunoassay Handbook (D. Wild, ed.,Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson,ed., Academic Press, 1996); Methods of Immunological Analysis (R.Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlagsgesellschaft mbH, 1993), Harlow and Lane Using Antibodies: A LaboratoryManual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1999; and Beaucage et al. eds., Current Protocols in Nucleic AcidChemistry John Wiley & Sons, Inc., New York, 2000).

As used herein, the term “derivative” in the context of anon-proteinaceous derivative refers to a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl, amine group, esterification,alkylation or phosphorylation, immobilization or addition of a polymer.

As used herein, the term “polymer” refers to compounds comprising threeor more monomeric units which may be the same or different. Thus,“polymer” refers to high molecular weight and/or insoluble polymers aswell as low molecular weight and/or soluble oligomers.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., amonkey, such as a cynomolgous monkey, and a human), and more preferablya human. In a preferred embodiment, the subject is a human. In a oneembodiment, the term “subject” excludes those subjects who suffer fromor have been diagnosed with secretory (acute) diarrhea.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe prevention, reduction, amelioration or elimination of inflammationor pain locally in the intestine, or to the prevention, reduction,amelioration or elimination of a symptom of an inflammatory boweldisease, or to the treatment or prevention of colon cancer or FAP.

The term “prevention, reduction, amelioration or elimination ofinflammation or pain or a symptom of inflammatory bowel disease” in thecontext of the present invention refers to at least one of thefollowing: prevention of inflammation, pain or the inflammatory boweldisease before it occurs, for example, in patients that suffered in thepast from inflammation or pain or the inflammatory bowel disease but arenow in a period of remission; elimination inflammation or pain or ofestablished inflammatory bowel disease; reduction of an undesiredsymptom of the disease as manifested by a decrease in the severity of anexisting condition; elimination or reduction of one or more medicationsused in treating the subject. Any amount of reduction in the severity ofa symptom, even if some of the symptom remains at a lower, moreacceptable level (“management”), is encompassed by the term hereindefined.

Since inflammation of the intestines and inflammatory bowel diseases areoften accompanied by other symptoms, such as abdominal discomfort, pain,bloating, fatigue, sleep disturbances, sexual dysfunction, headache,fibromyalgia (muscle aching), dyspepsia (upper abdominal discomfort orpain), chest pain, urinary or gynecological symptoms, anxiety anddepression. Reduction in at least one of these symptoms is alsoencompassed by the term “prevention, reduction, amelioration, managementor elimination of inflammation, pain or of a symptom of inflammatorybowel disease.”

As used herein, the term “therapeutically effective amount” refers tothat amount of the therapeutic agent sufficient to result in thetreatment of inflammation, pain or an inflammatory bowel disease, toprevent advancement of inflammation, pain or an inflammatory boweldisease, cause regression of inflammation, pain or an inflammatory boweldisease, or to enhance or improve the therapeutic effect(s) of anothertherapeutic agent administered to treat or prevent inflammation, pain oran inflammatory bowel disease. The term “therapeutically effectiveamount” also refers to that amount of the therapeutic agent sufficientto result in the treatment or prevention of colon cancer or FAP.

The following series of Examples are presented for purposes ofillustration and not by way of limitation on the scope of the invention.

EXAMPLES Example 1: Preparation of Pharmaceutical Formulations

Described below are illustrative methods for the manufacture andpackaging for different preferred pharmaceutical formulations of theproanthocyanidin polymer composition from C. lechleri according to thepresent invention.

IA. Encapsulated Enteric Coated Beads

Detailed descriptions of the batch formula and methods used to preparethe encapsulated enteric coated proanthocyanidin polymer compositionbead formulation based on sugar spheres are provided below. Eachhard-shell gelatin capsule contained 250 mg proanthocyanidin polymercomposition enteric coated beads. Capsules were packaged in HDPE bottlescontaining sixteen (16) 250 mg caps each. The formulation for entericcoated proanthocyanidin polymer composition beads contained 17.3% (w/w)of nonpareil seeds (sugar spheres 40/60 mesh, Paulaur, lot #60084060),64.5% proanthocyanidin polymer composition from C. lechleri, 1.5%hydroxypropylmethylcellulose (Methocel E5 Premium, Dow Chemical Co., lot#MM9410162E), 0.5% Opadry Clear (Colorcon, lot #S83563), 14.5%“EUDRAGIT™ L 30D” (Rohm Tech., lot #1250514132), 1.45% triethyl citrate(Morflex, lot #N5X291), glyceryl monostearate (Imwitor-900, Rohm Tech,lot #502-229), and purified water (USP).

The layering coating solution containing the proanthocyanidin polymercomposition was prepared by adding hydroxypropylmethylcellulose and theproanthocyanidin polymer composition to purified water (USP) and mixinguntil dissolved. The nonpareil seeds were loaded into the product bowlof the fluid bed processor (NiorPrecision Coater). The polymer solutionwas then layered on the nonpareil seeds by spraying the solution ontothe fluidized nonpareil seeds at a target bed temperature of 30-35° C.Once the proanthocyanidin polymer layering had been completed, a sealcoat using Opadry Clear (preparing by mixing the Opadry Clear withPurified Water, USP) was applied with a target bed temperature of 30-35°C. After the seal coat was applied, the pellets were discharged andscreened through 1000μ and 425μ screens, and the layered spheres largerthan 425μ and smaller than 1000μ were charged back into the fluid bedprocessor. Meanwhile, the enteric coating solution was prepared bymixing triethyl citrate and glyceryl monostearate to water that had beenheated to 65° C. and then mixing this solution with the “EUDRAGIT™ L30D-55”. The resulting enteric coating solution was then sprayed ontothe layered spheres in the fluidized bed processor, at a bed temperatureof 30-35° C., until all the enteric coating solution was layered on thebeads. Based on the results of the HPLC assay indicating that theproanthocyanidin polymer composition was present at a concentration of52.9%, the enteric coated beads were hand filled into a Size #0 hardshell gelatin capsule to provide a 250 mg dosage and then packaged intoa suitable HDPE bottles with a heat induction lined cap.

TABLE 1 BATCH FORMULA Product: Proanthocyanidin Polymer Enteric CoatedBeads Batch Size: 578.0 gm Raw Material Amount Used Per Batch SugarNonpareil Spheres, NF (40/60) 100.0 gm Proanthocyanidin PolymerComposition 372.8 gm Hydroxypropylmethylcellulose E5, USP 8.7 gm(K29/32) Opadry Clear (YS-1-19025A) 2.9 gm “EUDRAGIT ™ L 30D-55” 279.4gm (30% solids) Triethyl Citrate, NF 8.4 gm Glycerol Monostearate 1.4 gmWater, USP (Removed during processing) 1284.8 gm

1B. Encapsulated Enteric Coated Beads

Described below are the formula and methods used to prepare encapsulatedenteric coated bead formulations that do not contain nonpareil sugarspheres. One formulation contains 83.3% w/w proanthocyanidin polymercomposition, 0.5% w/w Opadry clear, 14.5% w/w “EUDRAGIT™ L 30D-55”, 1.9%w/w triethyl citrate and a 0.34% glyceryl monostearate.

The beads were first seal coated with a 5% solution of Opadry clear in a16 liter aeromatic MP-1 fluidized bed processor with a 50 mm Wurstercolumn. The coating parameters for the application of the seal coatingwere an inlet temperature of 50° C. to 60° C., an outlet temperature of25° C. to 40° C., an air volume of 30 to 40 CMH, a spray rate of 6 to 12grams per minute, and an air pressure of 2.5 Bar. After the seal coatwas applied, the beads were discharged and screened for beads largerthan 425μ and smaller than 1000μ. The beads of appropriate size werethen charged back into the fluid bed processes for enteric coating. Foreach 1000 grams of proanthocyanidin polymer composition beads, anenteric coating suspension was prepared from 811.97 grams “EUDRAGIT™ L30D-55”, 24.36 grams triethyl citrate, 4.36 grams glyceryl monostearateand 248.55 grams purified water. This suspension was prepared by gentlystirring the “EUDRAGIT™ L 30D-55” suspension continually and, in aseparate container, suspending and homogenizing the triethyl citrate andtalc in purified water. The triethyl citrate/talc mixture was then addedto the “EUDRAGIT™ L 30D-55” suspension, and the resulting coatingdispersion stirred during the spraying process to avoid settling. Thebeads were then coated in the fluidized bed processor under thefollowing parameters: The inlet temperature was 42° C. to 47° C.; theoutlet temperature was 28° C. to 34° C.; the air volume was 30-40 CMH;the spray rate was 6-12 grams/minute; and the air pressure was 2.5 Bars.The resulting enteric coated beads were then filled into a size #0 hardshell gelatin capsule.

1C. Enteric Coated Granules and Powder Particles

Described below is a method for formulating the proanthocyanidin polymercomposition as enteric coated granules or powder (microspheres with adiameter of 300-500μ) in either hard shell gelatin capsules or suspendedin an oral solution. The proanthocyanidin polymer composition powderparticles are prepared by high-shear powder mixing of theproanthocyanidin polymer composition and hydroxypropylmethylcellulose ina high speed mixer/granulator. The proanthocyanidin polymer compositiongranules are prepared by spraying polyvinylpyrrolidone on the powder inthe high speed mixer/granulator so that the powder particles agglomerateto form larger granules. Using fluidized bed equipment, the granules orpowder are then covered with a seal coat of Opadry Clear (mixed withwater) and then coated with the enteric coating “EUDRAGIT™ L 30D”applied as an aqueous dispersion containing 30% w/w dry methacrylatepolymer substance, which is supplied with 0.7% sodium lauryl sulfate NF(SLS) and 2.3% polysorbate 80 NF (Tween™ 20) as emulsifiers, to whichthe plasticizers, triethyl citrate and glyceryl monostearate, are addedto improve the elasticity of the coating. The final composition of theenteric coated powder is 81.8% w/w proanthocyanidin polymer composition,1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% w/w“EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate, and 0.25% w/w glycerylmonostearate. The final composition of the enteric coated granules is81.8% w/w proanthocyanidin polymer composition, 10%polyvinylpyrrolidone, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/wOpadry clear, 14.5% w/w “EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate,and 0.25% w/w glyceryl monostearate.

The enteric coated proanthocyanidin polymer composition granules orparticles may be filled into a hard shell gelatin capsule in an amountwhich provides a suitable dosage.

The enteric coated proanthocyanidin polymer composition granules orpowder particles can also be suspended in a solution for oraladministration, particularly for pediatric administration. Thesuspension solution is prepared by wetting 2 gramshydroxypropylmethylcellulose in 97.8 ml distilled water and 0.2 gramsTween™ 80; mixing this preparation to homogeneity by sonicating, heatingthe solution to 40° C. and stirring for three hours; and then adding theenteric coated proanthocyanidin polymer composition powder particles orgranules to the homogeneous solution.

1D. Enteric Coated Compressed Tablets

A method for formulating the proanthocyanidin polymer composition with adiluent as enteric coated tablets is described below. For each 350 mgtablet, 250 mg proanthocyanidin polymer composition is granulated with 7mg crosslinked sodium carboxymethylcellulose (“AC-DI-SOL™”) and asufficient mass of microcrystalline cellulose (“AVICEL™ PH 200/300”) tobring the total mass to 350 mg. These ingredients are mixed for 20 to 30minutes in a V blender. After the 20 to 30 minutes of mixing, 1.75 mgmagnesium stearate is added and the mixture is blended for an additional4 to 5 minutes. The resulting granules are compressed on a rotary tabletpress using 5/16th inch standard concave punches. The tablets are coatedwith an enteric coating mixture prepared from 250 grams “EUDRAGIT™ L 30D-55”, 7.5 grams triethyl citrate, 37.5 grams talc and 205 grams water.The tablets are then placed in a perforated pan coater (e.g. the“ACCELACOTA™” system) and rotated at 15 rpm at 40° C. The entericcoating formulation is sprayed using the following conditions: inlet airtemperature of 44° C.-48° C., exhaust air temperature of 29° C.-32° C.,product temperature of 26° C.-30° C., a 1 mm spray nozzle, a pan speedof 30 to 32 rpm, an airflow of 30-32 CFM, and a spray pressure of 20PSI. The tablets are finally cured for 30 minutes as the pan is rotatingat 15 rpm with an inlet air temperature of 60° C. and then, aftershutting off the heat, the tablets are rotated at 15 rpm until thetablets have cooled to room temperature.

1E. Enteric Coated Directly Compressed Tablets

A method for formulating the proanthocyanidin polymer compositionwithout a diluent as enteric coated tablets was carried out as describedbelow. Directly compressible proanthocyanidin polymer composition wasproduced according to the method described in Example 1F, infra. 125 mgtablets were prepared by blending 99.6% w/w directly compressibleproanthocyanidin polymer composition with 0.40% w/w magnesium stearatefor two minutes and then directly compressing the material into 125 mgtablets on a rotary press using ¼ inch diameter round standard concavepunches to a tablet hardness of 4-10 Kp.

The core tablets were tested and found to have an average hardness(n=10) of 4-10 Kp, friability (n=20) of less than 0.7%, an average tableweight (n=10) of 125 mg±7 mg, an average thickness (n=10) of 3.9 to 4.1mm, and a disintegration time (n=6) of not more than 20 minutes.

The coating dispersion was prepared by mixing, per 100 grams of tablets,22.22 grams of a 30% w/w “EUDRAGIT™ L 30D-55” suspension, kept gentlystirred with a mixture of 0.67 grams triethyl citrate, 1.67 grams talcand 20.44 grams purified water which had been mixed until homogeneous.The coating dispersion was continually stirred to avoid settling.

The tablets (in batches of 100,000) were coated with the coatingdispersion in a Compu-Lab 24 inch/30 L pan. The tablets were jogged inthe pan at a speed of 3-5 rpm and pre-warmed to a temperature of 35° C.to 40° C. The tablets were then coated with the enteric coatingdispersion to a 6% to 8% weight gain with the following parameters: aninlet temperature of 45° C. to 65° C.; an exhaust air temperature of 27°C. to 34° C.; a product temperature of 28° C. to 32° C.; a pan speed of8-14 rpm; an air flow of 180 to 240 CHM; an air spray pressure of 10-20psi (pounds per square inch); an initial spray rate of 3 to 4grams/min/kg; and a final spray rate of 4 to 8 grams/min/kg. The tabletswere then cured for 30 minutes in the pan with an inlet temperature of45° C. to 50° C. and a pan speed of 3 to 5 rpm. Finally, the tabletswere allowed to cool to room temperature in the pan at a pan speed of 3to 5 rpm. Four of the 125 mg tablets were then filled into a size zero,opaque Swedish orange-colored gelatin capsule.

The enteric coated proanthocyanidin polymer composition tablets weretested for content uniformity, drug release, microbiological tests andstability, and some analytical in process tests were also performed. Instability studies, the proanthocyanidin polymer composition remainedstable after six months of storage at room temperature as well as underaccelerated temperature and humidity conditions. Finally, the coretablets were tested and found to have an average hardness (n=10) of 4-10Kp, friability (n=20) of less than 0.7%, an average tablet weight (n=10)of 125 mg±7 mg, an average thickness (n=10) of 3.9 to 4.1 mm, and adisintegration time (n=6) of not more than 20 minutes.

1F. Enteric Coated Directly Compressed Tablets

Formulation of the proanthocyanidin polymer composition, without adiluent, as enteric coated tablets was carried out as described below.The directly compressible proanthocyanidin polymer composition wasisolated as described in Example 2, infra. The core tablets wereprepared by milling 250 mg proanthocyanidin polymer composition pertablet (approximately 16 kg total) in a Quadro Comil with an 024R (30mesh) screen and then blending the milled composition in a PattersonKelley 2 cubic foot twin shell blender. 1 mg magnesium stearate(Spectrum Quality Products, Inc., New Brunswick, N.J.) per tablet wasthen added to the composition in the blender and blended for 2 minutes.The blend was then compressed into 251 mg tablets (containing 250 mgproanthocyanidin polymer composition) on a rotary tablet press to atablet hardness of 8-15 Kp and friability less than 0.5%.

The coating dispersion was prepared by first mixing in a first containerthe 25 g (7.5 g solids) “EUDRAGIT™ L 30 D-55” (Huls America, Inc.,Somerset, N.J.) (weight given per 115 grams coated tablets) dispersion.The pigment dispersion was prepared by adding sequentially with constantstirring in a second container 39.59 g purified water, 3.30 grams talc(Alphafil™ 500) (Whittaker, Clark & Daniels, Inc., South Plainfield,N.J.), 6.06 g (3.15 g solids) White Dispersion (pigment)(Warner-Jenkinson, Inc., St. Louis, Mo.), and then 1.05 g triethylcitrate (Morflex, Inc., Greensboro, N.C.). The mixture was thenhomogenized for 15 minutes or until homogenous. While slowly stirring,the pigment dispersion was added to the “EUDRAGIT™ L 30 D-55” dispersionand then stirred for 30 minutes before spraying. Stirring was alsomaintained during the spraying process to avoid settling.

The tablets were coated in batches of 50,000 in a Compu-Lab 24 inch/30 Lpan with the following settings: 10-20 psi atomizing air pressure; 35°C.-60° C. pan inlet air temperature; 5 to 6 inches nozzle tip to tabletbed distance; and 4/2 baffles/nozzles. After adding the tablets to thepan, the pan was jogged at a speed of 3 to 5 rpm and heated to 40° C.The tablets were then sprayed to a weight gain of 11 to 13% with thefollowing parameters: 27°-33° C. target exhaust temperature (to beachieved within ten minutes of spraying); pan speed of 8 to 12 rpm;180-240 CFM air flow; and a spray rate of 2-5 g/min/kg. After achievingthe desired weight gain, the heat was shut off and the pan jogged at 3-5rpm until the tablets were cooled to below 30° C.

The tablets were encapsulated in size AA opaque Swedish orange coloredDB gelatin capsules (Capsugel, Greenwood, S.C.).

500 mg tablets were also produced as described above, except thatcoating was done on batches of 25,000 tablets to a weight gain of 8 to10%.

Example 2: Isolation of Directly Compressible Proanthocyanidin PolymerComposition

A directly compressible proanthocyanidin polymer composition (used toprepare the formulations in Examples 1E and 1F above) was isolated fromthe latex of the Croton lechleri plant as follows:

460 liters of Croton lechleri latex was mixed with 940 liters purifiedwater for ten minutes and then allowed to stand overnight (12 hours) at4° C. The red supernatant was pumped into a holding tank and the residuediscarded. The supernatant was then extracted with 200 liters n-butanolby mixing for ten minutes and then allowing the phases to separate. Then-butanol phase was discarded, and the aqueous phase was extracted twomore times with 200 liters n-butanol each time. After extraction, theaqueous phase was concentrated by ultrafiltration using a 1 kD cut-offmembrane (a low protein binding cellulose membrane), and then theretentate was dried in a tray dryer at approximately 37° C. (+2° C.).

For purification by column chromatography, 6 kg of the dried extract wasdissolved in 75 liters of purified water and stirred for 90 minutes. Thedissolved material was chromatographed on a two column chromatographysystem consisting of a 35 liter CM-Sepharose column (a weak cationexchange resin) and a 70 liter LH-20 column (a size-exclusion resin)connected in series. The material was loaded onto the CM-Sepharosecolumn, washed with 140 liters purified water, and then eluted onto theLH-20 column with 375 liters of 30% acetone. At this point, the twocolumns were disconnected, and the proanthocyanidin polymer compositionwas eluted from the LH-20 column with 250 liters of 45% liters acetone.Fractions were collected into 10 liter bottles and monitored with a UVdetector at 460 nm. Fractions containing material having detectableabsorbance at 460 nm were pooled and concentrated by ultrafiltrationusing a 1 kD cut-off membrane (a low protein binding cellulosemembrane). The retentate was dried using a rotary evaporator in awaterbath at approximately 37° C. (±2° C.).

The proanthocyanidin polymer composition was tested for directcompressibility. 250 mg portions of the proanthocyanidin polymercomposition, in the absence of any binders or excipients, was placedinto a tableting machine and then pressed into tablets of varyingthicknesses (i.e., the greater the pressure on the composition to formit into a tablet, the thinner the resulting tablet). The hardness of thetablets was then determined in a conventional hardness tester. Thefriability of tablets having a hardness of 8-15 kp was determined asdescribed in USP 23<1216>. The friability was less than 0.5% loss inweight.

Example 3: Components, Composition, and Manufacturing of a Drug Product

3A. Drug Product

The drug product, crofelemer, consists of an enteric-coated 125 mgtablet(s) over-encapsulated in a Size 00, opaque Swedish orange gelatincapsule and backfilled with microcrystalline cellulose. Each capsulecontains 1, 2, or 4 enteric-coated tablets. The tablet core consists of99.93% crofelemer and 0.07% magnesium stearate and is coated with amethacrylic acid copolymer.

3B. Components of Drug Product

Crofelemer is supplied by Napo Pharmaceuticals, Inc., and ismanufactured under current Good Manufacturing Practices (cGMP).Magnesium stearate is manufactured by Mallinckrodt (or equivalent) andmeets the specifications for magnesium stearate as described in 27USP/NF. The magnesium stearate is certified by the manufacturer to bederived from vegetable sources. Microcrystalline cellulose ismanufactured by FMC (or equivalent) and meets the specifications formicrocrystalline cellulose as described in 27 USP/NF. Both high-densityand low-density microcrystalline cellulose are employed. Methacrylicacid copolymer is manufactured by DeGussa under the trade name Eudragit®(L30-D55) and meets the specifications for methacrylic acid copolymer,Type C, as described in 27 USP/NF. Triethyl citrate is manufactured byMorflex (or equivalent) and meets the specifications for triethylcitrate as described in 27 USP/NF. Talc is manufactured by Whittaker,Clark and Daniels (or equivalent) and meets the specifications for talcas described in 27 USP/NF. Purified water is supplied by the drugproduct manufacturer and meets the specifications for purified water asdescribed in 27 USP/NF. Swedish orange opaque, Size 00, hard gelatincapsule bodies and caps are supplied by Capsugel, Inc. (Greenwood,S.C.). The manufacturer certifies that the capsules are made fromgelatins that meet the current National Formulary (NF) requirements forgelatin under cGMP.

3C. Composition of Drug Product

The composition of the tablet cores and the enteric-coated tablets isdescribed in Table 2 and Table 3, respectively. The amount of crofelemerand magnesium stearate is adjusted based on the anhydrous potency of thedrug substance, which corrects for the moisture content of thecrofelemer. The amount of weight gain after coating is approximately10%. The clinical batch size ranges from 100,000 to 150,000enteric-coated tablets. Subsequently, 1, 2, or 4 enteric-coated tabletsare placed in a Size 00 capsule and backfilled with microcrystallinecellulose to match weights of each capsule strength and placebo.

TABLE 2 Composition of Drug Product 125 mg Tablet Cores QuantitativeTheoretical Ingredient Grade Purpose Composition mg/unit dose CrofelemercGMP Active 99.93%  125 mg Magnesium 27 USP/NF Lubricant 0.07% 0.13 mgstearate Total  100% 125.13 mg

TABLE 3 Composition of Drug Product Enteric-Coated 125 mg TabletsQuantitative Theoretical Ingredient Grade Purpose Composition mg/unitdose Crofelemer cGMP Active 90.0%  125.13 mg tablet Eudragit 27 USP/NFCoating 7.4% 34.5 mg (10.4 L-30 D55 mg solids) Triethyl citrate 27USP/NF Plasticizer 0.8% 1.05 mg Talc 27 USP/NF Dispersing 1.8% 2.6 mgPurified water* 27 USP/NF Solvent N/A* 21.9 mg Total 100%  136.4 mg*Purified water is removed during process.

3D. Method of Manufacturing the Drug Product

I. Manufacture of Tablet Core

A sufficient amount of crofelemer and magnesium stearate, based on thepotency of crofelemer, on an anhydrous basis that adjusts for the amountof moisture, is staged prior to manufacture. Crofelemer is added to theblender and magnesium stearate is screened and added to the crofelemer.The crofelemer and magnesium stearate are blended, a representativeblend sample is taken, and the yield is determined. Yield must bebetween 100±3%. Blend uniformity is not determined, except as needed,because the blend is 99.9% active. The blend is directly compressed on arotary tablet press, using round concave punches. Finished tablet coresare de-dusted and placed in a container to await coating. Prior to thestart of the manufacturing run, pre-production runs are performed toadjust the speed and compression of the press in order to meet thetargeted tablet-core weight, thickness, and hardness. In addition,friability and disintegration are measured. During the manufacturingrun, tablet-core samples are taken at periodic intervals to ensure thatthe tablets continue to meet the targeted tablet weight, thickness, andhardness. Representative tablet cores are taken during the beginning,middle, and end of the production run for additional testing forhardness, thickness, weight, friability, and disintegration. The averagetablet-core weight must be within ±5% of the targeted tablet-coreweight. The number of tablet cores, sample tablet cores, tablet-corewaste, and blend waste are reconciled and the percent accountabilitycalculated. The percent accountability must not be less than 95% and notmore than 103%. A schematic of the tablet core manufacturing process isillustrated in FIG. 7

II. Coating of Tablet Core

The amount of Eudragit®, triethyl citrate, talc, and purified water iscalculated and staged based on a nominal tablet-core weight gain of 10%.Purified water is charged into a suitable container equipped with ahigh-shear mixer. The triethyl citrate and talc are added to thecontainer and mixed until homogenized. In a separate container equippedwith a propeller mixer, the Eudragit® dispersion is charged and mixed.The triethyl citrate and talc dispersion is added to the Eudragit®dispersion and is continuously stirred throughout the spraying process.The pan-coater machine parameters are adjusted as appropriate and thelines are charged with the coating dispersion. The tablet cores arecharged in a coating pan and warmed to 35 to 40° C. while jogging thecoating pan. Once at temperature, the average weight of the tablet coresis recorded and the targeted coated tablet weight is calculated.Subsequently, the tablet cores are sprayed and periodicallyweight-checked until the targeted weight gain is met. The targeted sprayrate (4 to 8 g/min/kg of tablet cores), the targeted exhaust temperature(35 to 40° C.), and inlet temperature are monitored at frequentintervals. The tablets are cured for 30 minutes at 45 to 50° C., andthen cooled. Representative enteric-coated samples are taken fortesting. The average enteric-coated tablet weight must be within ±5% ofthe targeted enteric-coated tablet weight.

The weight of enteric-coated tablets, enteric-coated tablet samples, andtheoretical quantity of enteric-coated tablets are determined and thepercent accountable yield calculated. The percent accountable yield mustnot be less than 95% and not more than 103%. The tablet-corespray-coating process is illustrated in FIG. 8

III. Manufacture of Over-Encapsulated Enteric-Coated Tablets

The enteric-coated tablets and microcrystalline cellulose are stagedseparately for the over-encapsulation of each capsule strength. Theamount of microcrystalline cellulose to be encapsulated is calculated inorder to achieve a nominal 600 to 800 mg capsule weight, dependent uponthe exact dosage form (125 mg, 250 mg, or 500 mg). The average weight ofthe capsules is calculated based on an average of 100 capsules. Thecapsules are filled using a semi-automatic over-encapsulation machinefitted with Size 00 change parts and adjusted to deliver the properamount of tablets and microcrystalline cellulose. Pre-production runsare performed in order to adjust the tray fill weight, the number ofturns on the tamper, and the number of times tamped in order to meet thetargeted gross capsule weight (capsule shell plus tablets andmicrocrystalline cellulose). Each tray is prepared by placing thecapsule bodies in the capsule tray. Each capsule body is filled with thetargeted amount of tablets and microcrystalline cellulose to achieve thetargeted fill. The caps are placed on the bodies and closed. Thecapsules are removed from the tray and de-dusted. A composite ofcapsules from each tray is collected and weighed for in-process andrelease testing. The average capsule weight is within ±5% of thetargeted capsule weight. The process is then repeated until the desirednumber of capsules is filled. Damaged capsules, enteric-coated tabletwaste, and microcrystalline cellulose waste are collected for finalproduct reconciliation. A composite of the finished capsules is sent forrelease testing. The number of finished capsules, sample capsules,damaged capsules, and drug substance waste are reconciled and thepercent accountability is calculated. The percent accountability mustnot be less than 95% and not more than 103%. A schematic of theover-encapsulation of the enteric-coated tablets is presented below.

Example 4: Investigation of Crofelemer Mechanism of Action

To further investigate the mechanism of action of crofelemer, crofelemerat 10 and 100 μg/mL, was evaluated in a selected panel of cellularcytokine release assays (IL-1α; TNF-α-induced and IL-1-induced PGE2release; IFN-γ; IL-2, IL-4; IL-5; IL-6; IL-8; IL10 and TNF-α) andmolecular assays (COX-1 and COX-2 enzyme assays, and glucocorticoid,serotonin 5HT3, δ-opiod, κ-opiond, μ-opiod, and non-selective opiodreceptor binding assays. Crofelemer was also tested in cytotoxicityassays corresponding to the ConA- and LPS-induced cytokine releaseassays as well as those corresponding to the TNF-a- and IL-1-induced PGErelease assays.

At both 10 and 100 μg/mL, crofelemer caused a 73% and 100% inhibition inthe COX1 and COX2 enzyme assays, respectively.

Further, crofelemer exhibited significant (>50%) inhibition in theIFN-y; IL2, IL-4; IL-6; IL-8; IL-10 and TNF-α cytokine release assays aswell as in the TNF-α-induced and IL-1-induced PGE2 release assays at 10and 100 μg/mL (i.e., all cytokine release assays with the exception ofIL-5). Crofelemer also displayed significant cytotoxic activity in theConA and IL-1-induced PGE2 cytotoxicity assays suggesting that in theConA-mediated cytokine release assays (IFN-γ, IL-2, IL-4 and IL-10) andthe IL-1-induced PGE2 release assay may be due to general cytotoxicity.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled. Such modifications areintended to fall within the scope of the appended claims.

All references, patent and non-patent, cited herein are incorporatedherein by reference in their entireties and for all purposes to the sameextent as if each individual publication or patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety for all purposes.

What is claimed is:
 1. A method of treating pain associated withinflammatory bowel disease comprising administering an extract from aCroton species or Calophyllum species to a subject in need thereof,wherein the dosage of the extract is bioequivalent to 250 mg to 500 mgof crofelemer per day and the inflammatory bowel disease is Crohn'sdisease or ulcerative colitis.
 2. The method of claim 1, wherein thesubject experiences an increase in the number of pain-free days.
 3. Themethod of claim 1, wherein the dosage is 250 mg to 500 mg of crofelemerper day.
 4. The method of claim 3, wherein the subject experiences anincrease in the number of pain-free days.
 5. The method of claim 1,wherein the dosage is administered twice per day.
 6. The method of claim1, wherein the extract is in a tablet form and the tablet has an entericcoating.
 7. The method of claim 1, wherein the extract is in a granularor powder form and particles of the granular or powder form have anenteric coating.
 8. A method of treating inflammation associated withinflammatory bowel disease comprising administering an extract from aCroton species or Calophyllum species to a subject in need thereof,wherein the dosage of the extract is bioequivalent to 250 mg to 500 mgof crofelemer per day and the inflammatory bowel disease is Crohn'sdisease or ulcerative colitis.
 9. The method of claim 8, wherein thelevel of inflammation in the subject is decreased by at least 20%. 10.The method of claim 8, wherein the dosage is 250 mg to 500 mg ofcrofelemer per day.
 11. The method of claim 10, wherein the level ofinflammation in the subject is decreased by at least 20%.
 12. The methodof claim 8, wherein the dosage is administered twice per day.
 13. Themethod of claim 8, wherein the extract is in a tablet form and thetablet has an enteric coating.
 14. The method of claim 8, wherein theextract is in a granular or powder form and particles of the granular orpowder form have an enteric coating.